The blood of vertebrates is contained within a closed circulatory system. Partially as a means of maintaining this closed system, a mechanism has evolved for sealing this system in the event of injury. One component of vertebrate blood is a kind of cell fragment called a thrombocyte or platelet. Certain dissolved proteins and the platelets are involved in blood-clotting, a complex series of reactions which occur in case of injury to the circulatory system. The end result of these reactions is the formation of a clot, which temporarily seals off the injured area until the damage is repaired.
The main reactions of the clotting process involve thrombokinase, an enzymatically active substance which is released from ruptured platelets. Thrombokinase begins a cascade of events by interacting with both prothrombin (which is inactive) and calcium ions in the blood to produce active thrombin. Thrombin acts on fibrinogen to form fibrin, an insoluable coagulated protein which then forms a meshwork of fibers (a clot) to prevent loss of blood.
However, in many instances, the formation of a clot may be detrimental when it is not repairing an injury. Formation of blood clots within a vessel can block blood flow in that vessel producing damage to the vessel, surrounding tissue, and to tissue served by the vessel. The presence of fibrin within the circulatory system of a host is an indication either of traumatic injury resulting in a lesion to the circulatory system, or pathological condition, having the potential for serious damage.
Currently diagnosis of the presence of blood clots is achieved by cardiovascular imaging techniques using a radio opaque contrast agent administered intravascularly. Current techniques detect clots by demonstrating the alteration of blood flow around a clot or by location of the clot directly. The flow-measuring techniques use a radio opaque contrast agent or colloidal Technetium. The present direct location techniques use .sup.125 I labeled fibrinogen which must be incorporated into the clot as fibrin. However, this direct technique is not 100% reliable, for example it does not reliably detect old clots. Also, the method is cumbersome to perform, requires 24 hours to provide results, interferes with other diagnostic tests and is ineffective for deep vein thrombosis, a clinically significant diagnostic problem.
In addition, numerous hosts are hypersensitive to the components of contrast agents, their prolonged residence time in the host interferes with other tests and there are numerous contraindications to their use.
As a counter mechanism to clot formation, blood plasma has an enzyme system which dissolves blood clots by catalysis of fibrin to soluble degradation proteins. The enzyme responsible for this transformation, which results in blood clot dissolution, is plasmin. Plasminogen, a precursor of plasmin, is converted to plasmin by enzymes termed plasminogen activators.
The known plasminogen activators include streptokinase, urokinase (u-PA) and a more recently discovered activator, tissue plasminogen activator (t-PA). Urokinase, the plasminogen activator found in urine, is available commercially as a fibrinolytic agent. However, its use has been somewhat limited because of its low specific activity and weak affinity for fibrin.
It has been claimed that a urokinase (u-PA) preparation may be labeled with a radioactive label and used as a diagnostic agent. Hausin, et al., U.S. Pat. No. 4,381,346. However, this use still suffers from many of the inherent defects of u-PA including potential adverse effects noted in u-PA's use as a fibrinolytic drug.
It has been discovered that t-PA has a rather high affinity for fibrin in vitro. Tissue-PA's high affinity for fibrin can be used to provide a diagnostic agent for locating blood clots.
Therefore, it is an object of this invention to provide a diagnostic reagent with high affinity for fibrin.
It is a further object of this invention to provide a diagnostic agent which can be administered systemically without causing adverse reactions.
It is a still further object of this invention to provide a diagnostic agent which will be rapidly cleared from the bloodstream to minimize interference with other diagnostic tests.
It is still a further object of this invention to provide a diagnostic agent to identify blood clots.